Abstract:

The majority of patients suffering from Alzheimer’s disease (AD) demonstrate cerebral vascular changes and impaired regulation
of cerebral blood flow, which has been assumed to play an important role in AD pathogenesis (vascular hypothesis of AD). There is
strong evidence that both β-amyloid (Aβ) oligomers and plaques contribute to vascular injuries and functional impairments of the
neurovascular unit. Vice versa, Aβ lesions can be triggered by hypertension and ischemic brain injury, while Aβ aggregates appear to
have anti-angiogenic properties. Cholinergic dysfunction may result in impaired cerebral blood flow with consequences on normal function
of the neurovascular unit including processing of the amyloid precursor protein (APP).

To characterize in vivo the developmental relationship between Aβ formation and deposition, cortical cholinergic innervation and cerebrovascular
abnormalities, transgenic Tg2576 mice that overexpress the Swedish double mutation of human APP, and demonstrate significant
cerebral cortical deposition of Aβ plaques at ages from 9 months onwards, were considered as an appropriate animal model. Using
the somatosensory cortex as a representative region, serial cryocut sections, were obtained from mice at ages ranging from 4 up to 18
months. These were subjected to immunohistochemistry to label vascular endothelial cells (anti-glucose transporter 1 (GluT1) immunostaining),
cholinergic nerve terminals (anti-vesicular acetylcholine transporter (VAChT) immunostaining) and β-amyloid plaques
(thioflavin S, and/or Solanum tuberosum lectin staining). This was followed by a thorough quantitative evaluation of the age-related spatial
relationship between cerebral cortical capillaries, Aβ plaques and cholinergic terminals, using computer-assisted image analysis.

The density of cholinergic terminals estimated by evaluation of VAChT immunohistochemistry in somatosensory cortical sections of
wild type mice did not change with aging regardless of the cortical layer examined, while in cortical layers II/III and IV of somatosensory
cortex of transgenic Tg2576 mice age-related decreases in cholinergic fiber densities were assessed.

However, quantitative morphometric analysis demonstrated an age-related reduction in the number of varicosities on cholinergic fibers,
particularly in layer IV, in both transgenic Tg2576 mice and non-transgenic littermates.

Cholinergic innervation of microvessels in the somatosensory cortex decreased with aging in both Tg2576 mice and non-transgenic littermates,
as revealed by estimating the ratio of the number of cholinergic vascular contacts and total length of blood vessel. There was no
significant difference in the perivascular cholinergic innervation in areas that demonstrated significant plaque load and those with no
plaque deposits regardless of the cortical layer examined.

The density of blood vessels estimated in the somatosensory cortex of transgenic mice by anti-GluT-1 immunohistochemistry did not differ
to that obtained in wild type mice before the onset of plaque deposition (younger than 10 months). However, in aged, 18-month-old
Tg2576 mice, demonstrating high plaque loads, decreased blood vessel densities, particularly in layer IV of the somatosensory cortex,
were observed.

The data obtained in this study strongly support the idea of an age-related interplay between Aβ accumulation, cholinergic dysfunction,
and vascular impairments. However, it remains to be elucidated as to which processes play a causative role and which events are secondary.
A potential mechanism is provided by the vascular hypothesis of AD. Aging-, and life-style-associated damage of the brain microvasculature
may affect Aβ clearance and perivascular drainage, promoting cerebrovascular Aβ deposition, inducing partial loss of cholinergic
vascular innervation and changes in vascular function, angiogenesis and upregulation of the vesicular endothelial growth factor
(VEGF) with consequences on APP processing and Aβ accumulation.

Abstract:The majority of patients suffering from Alzheimer’s disease (AD) demonstrate cerebral vascular changes and impaired regulation
of cerebral blood flow, which has been assumed to play an important role in AD pathogenesis (vascular hypothesis of AD). There is
strong evidence that both β-amyloid (Aβ) oligomers and plaques contribute to vascular injuries and functional impairments of the
neurovascular unit. Vice versa, Aβ lesions can be triggered by hypertension and ischemic brain injury, while Aβ aggregates appear to
have anti-angiogenic properties. Cholinergic dysfunction may result in impaired cerebral blood flow with consequences on normal function
of the neurovascular unit including processing of the amyloid precursor protein (APP).

To characterize in vivo the developmental relationship between Aβ formation and deposition, cortical cholinergic innervation and cerebrovascular
abnormalities, transgenic Tg2576 mice that overexpress the Swedish double mutation of human APP, and demonstrate significant
cerebral cortical deposition of Aβ plaques at ages from 9 months onwards, were considered as an appropriate animal model. Using
the somatosensory cortex as a representative region, serial cryocut sections, were obtained from mice at ages ranging from 4 up to 18
months. These were subjected to immunohistochemistry to label vascular endothelial cells (anti-glucose transporter 1 (GluT1) immunostaining),
cholinergic nerve terminals (anti-vesicular acetylcholine transporter (VAChT) immunostaining) and β-amyloid plaques
(thioflavin S, and/or Solanum tuberosum lectin staining). This was followed by a thorough quantitative evaluation of the age-related spatial
relationship between cerebral cortical capillaries, Aβ plaques and cholinergic terminals, using computer-assisted image analysis.

The density of cholinergic terminals estimated by evaluation of VAChT immunohistochemistry in somatosensory cortical sections of
wild type mice did not change with aging regardless of the cortical layer examined, while in cortical layers II/III and IV of somatosensory
cortex of transgenic Tg2576 mice age-related decreases in cholinergic fiber densities were assessed.

However, quantitative morphometric analysis demonstrated an age-related reduction in the number of varicosities on cholinergic fibers,
particularly in layer IV, in both transgenic Tg2576 mice and non-transgenic littermates.

Cholinergic innervation of microvessels in the somatosensory cortex decreased with aging in both Tg2576 mice and non-transgenic littermates,
as revealed by estimating the ratio of the number of cholinergic vascular contacts and total length of blood vessel. There was no
significant difference in the perivascular cholinergic innervation in areas that demonstrated significant plaque load and those with no
plaque deposits regardless of the cortical layer examined.

The density of blood vessels estimated in the somatosensory cortex of transgenic mice by anti-GluT-1 immunohistochemistry did not differ
to that obtained in wild type mice before the onset of plaque deposition (younger than 10 months). However, in aged, 18-month-old
Tg2576 mice, demonstrating high plaque loads, decreased blood vessel densities, particularly in layer IV of the somatosensory cortex,
were observed.

The data obtained in this study strongly support the idea of an age-related interplay between Aβ accumulation, cholinergic dysfunction,
and vascular impairments. However, it remains to be elucidated as to which processes play a causative role and which events are secondary.
A potential mechanism is provided by the vascular hypothesis of AD. Aging-, and life-style-associated damage of the brain microvasculature
may affect Aβ clearance and perivascular drainage, promoting cerebrovascular Aβ deposition, inducing partial loss of cholinergic
vascular innervation and changes in vascular function, angiogenesis and upregulation of the vesicular endothelial growth factor
(VEGF) with consequences on APP processing and Aβ accumulation.